The focus of my lab has broadened to include both the study of secreted Frizzled-related proteins (sFRPs) and cellular responses to Wnt stimulation. Defining the specificity of Wnt/Frizzled(Fzd)/sFRP interactions continues to be one of our long-term goals. In the past, we purified four recombinant sFRPs and made progress in the isolation of three Wnt proteins. We also identified a cell line with a low background of Wnt/Fz expression, and separately introduced three epitope-tagged Fzds into these cells to assist our study of Wnt/Fz/sFRP binding and signaling. During the past year, we engineered epitope-tagged versions of the remaining Fzds family members, such that we now have a full set of ten Fzds with the same tag to facilitate our investigation of protein-protein interactions and signaling in various model systems. In particular, we have been focusing on an alleged association of sFRPs with Fzds, and the potential functional consequences of these interactions. Thus far, our results suggest that such sFRP-1/Fzd binding occurs and in some instances may activate non-canonical Wnt signaling. We have demonstrated for the first time that Wnt stimulation induces Ewing's sarcoma cells to form neurites, and have begun to define the mechanisms that account for this cellular response. Fzd3 is the primary Wnt receptor that mediates this event, which also involves the previously identified Wnt effector molecules, Dishevelled-2 and Dishevelled-3, and amino-terminal c-Jun kinase (JNK). Consistent with one of our general objectives (see above), we observed that Dickkopf-1 also promoted neurite outgrowth in Ewing's cells, apparently by activating similar non-canonical Wnt signaling. This experimental model should prove useful in understanding how Wnts facilitate the formation of cell-cell connections in the nervous system, and perhaps enhance nerve regeneration. This investigation also will provide insight about the properties of Ewing's sarcoma cells. A manuscript describing this work has been submitted for publication. Last year we initiated a collaboration to investigate the role of R-spondins in the stimulation of Wnt/beta-catenin signaling and tumorigenesis. Results during the current fiscal year suggested that R-spondins may signal through additional pathways to regulate gene expression and presumably other cellular responses. Various R-spondin2 derivatives have been generated to determine the structural requirements for R-spondin biological activities, in particular, regulation of gene expression. This research should provide new information about a hypothesized connection between Wnts, Rspondins and tumorigenesis.